Filtration methods and devices

ABSTRACT

A method of filtering a liquid sample that includes passing a sample comprising at least one biological organism through a filter membrane at a passive water volume flux of at least 10 L/m 2 ·h·psi, wherein the filter membrane comprises a Bubble Point pore size of no more than 1.0 μm, thereby retaining at least one biological organism on the surface of the membrane; and detecting the at least one biological organism retained on the surface of the filter membrane.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/352,205, filed Jun. 7, 2010, which is incorporatedherein by reference in its entirety.

This application has associated with it a sequence listing with the filename Sequence_Listing_(—)66415WO003, created May 25, 2011 and contains1,753 bytes, which is incorporated herein by reference.

BACKGROUND

Membrane filtration is a standard step in many methods of analyzing aliquid sample for the presence of biological organisms. Such analysesare commonly performed in the interest of, for example, food safety,water quality, and/or environmental monitoring and/or study. Manymembranes having an average pore size of 0.45 μm or less (e.g.,cellulose acetate, nylon, etc. membranes) may be able to trap bacteriaand allow growth of the trapped bacteria when placed on a suitablemedium. It can be difficult, however, to recover bacteria from suchmembranes. These membranes, despite having an average pore size of 0.45μm or less, typically possess a significant number of pores at themembrane surface that are larger than the biological organisms and,therefore, have torturous pore structure into which biological organismsmay become trapped.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of filtering aliquid sample. Generally, the method includes passing a samplecomprising at least one biological organism through a filter membrane ata passive water volume flux of at least 10 L/m²·h·psi, wherein thefilter membrane comprises a Bubble Point pore size of no more than 1.0μm, thereby retaining at least one biological organism on the surface ofthe membrane; and detecting the at least one biological organismretained on the surface of the filter membrane.

In certain embodiments, the biological organism may be detected in situon the filter membrane, while in other embodiments, the biologicalorganism may be removed from the filter membrane before being detected.Thus, in some embodiments, the method includes eluting retainedbiological organisms from the filter membrane.

In some embodiments, the method can further include quantifying at leastone of the biological organisms.

In some embodiments, the method can include detecting and/or quantifyingthe biological organism no more than 24 hours after the sample is passedthrough the filter membrane.

In some embodiments, the liquid sample can include, for example, foodsamples, environmental samples, or water samples.

In some embodiments, the filter membrane may be provided in functionalcommunication with an absorbent member.

In some embodiments, the method can include reducing the volume of theliquid sample by at least 50%.

In another aspect, the present invention provides a filter device.Generally, the filter device includes a pocket comprising a pocketsurface that defines a pocket volume; an absorbent member disposed on atleast a portion of the pocket surface; and a filter membrane disposed onat least a portion of the absorbent member in fluid communication withthe pocket volume.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. SEM image of R1933-18 (nascent, 0.23 μm) membrane. 1A Open side(5000×), 1B Tight side (5000×), 1C Cross-section (500×).

FIG. 2. SEM image of R1933-7 (nascent, 0.34 μm) membrane. 2A Open side(5000×), 2B Tight side (5000×), 2C Cross-section (500×).

FIG. 3. SEM image of R1933-8B (nascent, 0.51 μm) membrane. 3A Open side(5000×), 3B Tight side (5000×), 3C Cross-section (500×).

FIG. 4. SEM image of R1901-11 (nascent, 0.74 μm) membrane. 4A Open side(5000×), 4B Tight side (5000×), 4C Cross-section (500×).

FIG. 5. SEM image of PAN membranes (10,000×). 5A PAN-1 (0.613 μm), 5BPAN-2 (0.531 μm), 5C PAN-3 (0.367 μm).

FIG. 6. SEM images (10,000×) of MF-Millipore Type HAWP (0.4 μm) (6A) andIsopore polycarbonate filter (0.4 μm) (6B). Both membrane images are ofsides receiving sample.

FIG. 7. A perspective schematic view of one embodiment of a filtrationdevice.

FIG. 8. A schematic side view of one embodiment of a filtration device.

FIG. 9. A schematic side view of one embodiment of a filtration device.

FIG. 10A. A schematic side view of one embodiment of a filtrationdevice.

FIG. 10B. A schematic side view of one embodiment of a filtration devicewith a sample-retrieval port.

FIG. 11. An exploded side view of one embodiment of a filtration deviceaccording to the present disclosure.

FIG. 12. A plan view of the assembled filtration device of FIG. 11.

FIG. 13. A cross-sectional schematic side view of an embodiment of afiltration device with a support member, according to the presentdisclosure.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

We describe herein methods in which liquid samples may be analyzed forthe presence of one or more biological organisms. Depending upon thespecific context in which the methods are practiced, the methods caninvolve passing a liquid sample through a membrane having a Bubble Pointpore size of no more than 1.0 μm while still providing a relatively highwater volume flux.

Optionally, the method can further provide that a relatively highpercentage of the biological organisms of the sample are retained on thesurface of the membrane rather than being imbedded in pores of themembrane. Thus, in some embodiments, the method further provides that arelatively high percentage of the biological organisms retained on thesurface of the membrane may be easily recovered from the membranesurface. In other cases, the method can further provide that the volumeof the liquid sample is significantly reduced.

The following terms shall have the indicated meanings.

“Active” refers to filtration methods in which a mechanized force (e.g.,a vacuum) drives the movement of liquid sample through a filtermembrane.

“Biological analyte” refers to a molecule, or a derivative thereof, thatoccurs in or is formed by an organism. For example, a biological analytecan include, but is not limited to, at least one of an amino acid, anucleic acid, a polypeptide, a protein, a polynucleotide, a lipid, aphospholipid, a saccharide, a polysaccharide, or any combination of twoor more thereof. Exemplary biological analytes can include, but are notlimited to, a metabolite (e.g., staphylococcal enterotoxin), an allergen(e.g., a peanut allergen), a hormone, a toxin (e.g., Bacillus diarrhealtoxin, aflatoxin, etc.), RNA (e.g., mRNA, total RNA, tRNA, etc.), DNA(e.g., plasmid DNA, plant DNA, etc.), a tagged protein, an antibody, anantigen, or any combination of two or more thereof.

“Bubble Point pore size” refers to a computed average pore size of amembrane. Bubble Point pore size is based on the fact that liquid isheld in the pores of a filter by surface tension and capillary forces.The minimum pressure required to overcome surface tension and forceliquid out of the pores is a measure of the pore diameter. The formulafor computing Bubble Point pore size is:

$D = \frac{4\sigma \; \cos \; \theta}{P}$

where:P=bubble-point pressure;σ=surface tension of the liquid (72 dynes/cm for water);θ=liquid-solid contact angle (which for water is generally assumed to bezero); andD=diameter of the pore.

“Elute” and variations thereof refer to removing biological organismsfrom a filter membrane using low stringency physical methods such as,for example, gravity, manual shaking, or vortexing.

“Entrapped” refers to biological organisms captured by a filter membranethat are not easily eluted from the filter membrane because, forexample, the biological organisms are captured in spaces within themembrane.

“Passive” refers to filtration in which no mechanized force (e.g., avacuum) drives the movement of liquid sample through a filter membrane.Passive filtration methods include filtration using, for example,gravity and/or absorption of fluid by an absorbent to drive the movementof fluid through a filter membrane.

“Recovered” refers to biological organisms that are eluted from a filtermembrane in condition for detection and/or further analysis.

“Retained” and variations thereof refer to biological organisms that aredisposed on the filter membrane surface after filtration and are easilyeluted from the filter membrane.

“Water volume flux” refers to a volume of fluid passing through a unitarea of membrane per unit time per unit of pressure. Unless otherwiseindicated, water volume flux is expressed herein as liters of liquidsample passing through one square meter of membrane per hour per poundper square inch of pressure (L/m²·h·psi).

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

Many commercially available membranes are used for filtering andremoving biological organisms from liquid samples. The art of membranefiltration is well known. Existing commercially available membranes,however, typically have a torturous path and possess significant amountof large pores at the surface. This feature makes it difficult torecover filtered biological organisms because the captured biologicalorganisms may become lodged in the pores of the filter membrane and are,therefore, difficult to remove from the filter intact so that thecaptured biological organisms may be analyzed.

Here, we describe methods in which liquid samples may be filtered usinga filter membrane so that each of two competing parameters aresatisfied. First, the methods involved retaining biological organisms onthe surface of the filter membrane so that the retained biologicalorganisms may be easily eluted from the membrane for further analysis.However, existing methods designed to capture high percentages ofbiological organisms do so by using membranes having very small averagepore size. This necessarily limits the water flux volume and,consequently, the rate at which a given volume of liquid sample may beprocessed. Thus, the methods described herein further provide a greaterwater flux volume than presently observed in filtration methods.

In some cases, the method can involve filtering large volumes (e.g.,multiple liters) of liquid sample such as may be desired for, forexample, environmental testing, water quality testing, water treatmenttesting, and/or testing of repaired and/or restored water utility pipes.For example, using present methods for testing the water quality inrepaired and/or restored water pipes, it can take two to three days toconfirm that the water quality is sufficient to restore water service.Using the methods described herein, however, it may be possible toconfirm water quality rapidly enough that water service can be restoredwithin 24 hours.

In other cases, the method can involve filtering enriched food samples,food processing water samples, and/or potable water samples.

The methods described herein can decrease the time required for suchtesting in at least two ways. First, the methods permit large volumes ofliquid sample to be filtered and analyzed. Second, the methods result ina significant percentage of the captured biological organisms beingretained on the surface of the filter membrane so that they are morereadily recovered by elution.

In other applications, the methods can involve relatively rapidfiltration of smaller volumes of liquid samples (e.g., less than oneliter). In these applications, too, the combination of relatively highwater flux volume and retaining biological organisms on the filtermembrane for easy recovery promotes more rapid and/or simpler filtrationof liquid samples. In some of these applications, biological organismsmay be recovered using simple gravity to dislodge retained biologicalorganisms from the filter membrane. In other applications, a liquidsample may be significantly concentrated—i.e., some quantity less thanthe entire liquid volume may be passed through the filter membrane.Because a significant percentage of the biological organisms areretained on the filter membrane rather than being entrapped within thefilter membrane, a greater percentage of the biological organisms in theoriginal sample may be recovered in the remaining liquid volume, therebyconcentrating the biological organisms for further identification and/orother analyses.

Generally, the methods include passing a sample comprising at least onebiological organism through a filter membrane having a Bubble Point poresize of no more than 1.0 μm, thereby retaining at least one biologicalorganism on the surface of the membrane. The methods include passing thesample through the filter membrane at a water volume flux of at least 10L/m²·h·psi for passive filtration, or a water volume flux of at least100 L/m²·h·psi for active filtration. The method further includesdetecting the at least one biological organism retained on the surfaceof the filter membrane.

The liquid sample may be obtained from any suitable source, and mayinclude a water sample. Exemplary water samples include environmentalsamples (e.g., lakes, rivers, streams, oceans, ponds, etc.), waterutility/water treatment samples (e.g., water supply pipes, watertreatment facilities, water treatment discharge, sewage, etc.), potablewater samples (e.g., bottled water, well water) or food samples (e.g.,liquid foods, food samples processed by, for example, homogenizing,etc.). Samples may be filtered as collected or may be processed to somedegree prior to filtration and further analysis.

The biological organism may be any prokaryotic or eukaryotic organismfor which detection and/or quantitation in a liquid sample may bedesired. Accordingly, the biological organism may include, for example,a parasite, or a microbe such as, for example, a unicellular eukaryoticorganism (e.g., a yeast), an algae, or a bacterium. Exemplary microbesinclude, for example, coliform bacteria. Exemplary bacterial speciesinclude, for example, Escherichia spp. (e.g., E. coli), Enterobacterspp. (e.g., E. aerogenes) Enterococcus spp., (e.g., E. faecalis),Citrobacter spp., (e.g., C. freundii), Klebsiella spp., Shigella spp.,Salmonella spp. (e.g., S. enterica), Listeria spp, Pseudomonas spp.,etc.

The filter membrane may possess a Bubble Point pore size of no more than1.0 μm, although the methods may be performed using a filter membranehaving a Bubble Point pore size of greater than 1.0 μm. Exemplary filtermembranes can have a Bubble Point pore size of, for example, no morethan 0.95 μm, no more than 0.9 μm, no more than 0.85 μm, no more than0.8 μm, no more than 0.75 μm, no more than 0.7 μm, no more than 0.6 μm,or no more than 0.5 μm. Suitable Bubble Point pore sizes may bedetermined, at least in part, by, for example, the size of biologicalorganism that is desired to be detected, the volume of sample to befiltered, and the depth of the filter membrane's pores. In the contextof multi-zone membranes, The Bubble Point pore size is measured for thezone positioned to retain biological organisms.

Exemplary filter membranes can be made by, for example, TIPS (thermallyinduced phase separation) process, SIPS (solvent induced phaseseparation) process, VIPS (vapor induced phase separation) process,stretching process, track-etching, or electrospinning (e.g., PAN fibermembranes). Suitable membrane materials include, for example,polyolefins (e.g., polyethylene and/or polypropylene),ethylene-chlorotrifluoroethylene copolymer, polyacrylonitrile,polycarbonate, polyester, polyamide, polysulfone, polyethersulfone,polyvinylidene fluoride (PVDF), cellulose ester, and/or combinationsthereof.

Suitable membranes may be characterized as porous membranes or asnanofiber membranes. Nanofiber filter membranes can have the fiberdiameter less than 5 μm such as, for example, less than 1 μm. Nanofibermembranes may be prepared from, for example, polyacrylonitrile,polyvinylidene fluoride, a cellulose ester, polyvinyl acetate, polyvinylalcohol, polyvinyl butyral, and/or combinations thereof.

Certain TIPS polyolefin membranes can be prepared so that they possess asingle, homogeneous zone of membrane structure, each zone having adifferent pore microstructure. In other cases, a TIPS membrane may beprepared as a multi-zone membrane that includes two or more zones, eachzone having a different pore microstructure. A multi-zone TIPS membranemay contain distinct zones or, alternatively, may possess a transitionzone between two otherwise distinct zones.

Exemplary filter membranes include membranes and methods for makingexemplary filter membranes are described in, for example, in U.S. Pat.No. 4,539,256, U.S. Pat. No. 4,726,989, U.S. Pat. No. 4,867,881, U.S.Pat. No. 5,120,594, U.S. Pat. No. 5,260,360, International PatentPublication No. WO2010/078234, International Patent Publication No.WO2010/071764, U.S. Provisional Patent Application Ser. No. 61/351,441,entitled, “Coated Porous Materials,” filed Jun. 4, 2010, and U.S.Provisional Patent Application Ser. No. 61/351,447, entitled, “Processfor Making Coated Porous Materials,” filed Jun. 4, 2010.

In some cases, active filtration can provide a water flux volume of atleast 100 L/m²·h·psi, although the methods may be performed at a waterflux volume less than 100 L/m²·h·psi. Exemplary water flux volume valuesusing active filtration include, for example, at least 250 L/m²·h·psi,at least 500 L/m²·h·psi, at least 750 L/m²·h·psi, at least 1000L/m²·h·psi, at least 1250 L/m²·h·psi, at least 1500 L/m²·h·psi, at least1750 L/m²·h·psi, at least 2000 L/m²·h·psi, at least 2500 L/m²·h·psi, orat least 3000 L/m²·h·psi. The maximum water flux rate may be determined,at least in part, by the maximum capacity of the mechanized force usedto drive movement of the liquid sample through the filter membrane, thestrength and/or durability of the filter membrane, and the Bubble Pointpore size of the filter membrane.

In some cases, passive filtration can provide a water flux volume of atleast 10 L/m²·h·psi, although the methods may be performed at a waterflux volume less than 10 L/m²·h·psi. Exemplary water flux volume valuesusing active filtration include, for example, at least 10 L/m²·h·psi, atleast 20 L/m²·h·psi, at least 25 L/m²·h·psi, at least 32 L/m²·h·psi, atleast 50 L/m²·h·psi, at least 60 L/m²·h·psi, at least 75 L/m²·h·psi, atleast 88 L/m²·h·psi, at least 95 L/m²·h·psi, or at least 100 L/m²·h·psi.The maximum passive water flux rate may be determined, at least in part,by the Bubble Point pore size of the filter membrane and/or the fluxgradient generated by, for example, an absorbent material positioned influid communication with the filter membrane so that it is capable ofdrawing at least a portion of the fluid sample through the filtermembrane.

In some embodiments, the method results in at least 30% of thebiological organisms in the sample are retained by the filter membrane,although the methods may be practiced so that fewer than 30% of thebiological organisms in the sample being retained by the filtermembrane. In exemplary methods, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least97.5%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, atleast 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least99.7%, at least 99.8%, or at least 99.9% of the biological organisms inthe sample are retained by the filter membrane.

As noted above, some embodiments can include, for example, a filtermembrane in functional communication with an absorbent member within alarger device. Embodiments of such devices 10 are shown in FIGS. 7-13.In such embodiments, an absorbent member 16 can generate a water fluxgradient sufficient to draw liquid across the filter membrane and intothe absorbent member 16. In some embodiments (e.g., as shown in FIG. 7),the filter membrane can cover at least a portion of the absorbent member16 so that flow of liquid into the absorbent member 16 requires that theliquid flow through the filter membrane. In this manner, biologicalorganisms in the liquid sample may be retained by the filter membrane.The device 10 further comprises a container 12 with an optional closure13. The container 12 may comprise a flexible, deformable container 12such as a ZIPLOK brand storage bag, for example, having a sealableclosure 13 comprised of interlocking components (13 a and 13 b).

In one particular embodiment, shown in FIG. 8, the absorbent member 16may be disposed on a surface of a pocket 18 formed in a device 10. Thepocket 18 may, therefore, function as a receptacle for the liquid sample24. The absorbent member 16 can optionally include a nonwoven backing 20to assist in forming a seal 26, thereby fastening the absorbent member16 to a portion of the container 12. The seal 26 may be formed via anadhesive or by heat-bonding, for example. The filter membrane 14 may bedisposed on the open-pocket surface (i.e., facing the interior volume ofthe pocket 18) of the absorbent member 16. In use, illustrated in FIGS.9 and 10A-B, the device 10 may be oriented so that the filter membrane14 is in a generally vertical orientation. In some cases, therefore,biological organisms retained by the filter membrane 14 may elute fromthe filter membrane 14 due to gravity. Thus, as liquid is absorbed bythe absorbent member 16, biological organisms in the liquid sample 24are concentrated. When the liquid sample 24 reaches a volumecorresponding to a desired level of concentration of the biologicalorganisms, a portion of the concentrated liquid sample 24 (FIG. 10A) maybe removed from the device 10 by using, for example, a syringe 22. Theremoved portion of the concentrated liquid sample may then be subjectedto further analysis.

In some embodiments, a portion of the concentrated liquid sample 24(FIG. 10B) may be removed from the device 10 by using, for example, asample port 30 to obtain the concentrated liquid sample 24. The sampleport 30 may include an elastically-deformable split septum 40 (e.g., asplit-cap TPE plug style cap available from Micronic North America, LLC;McMurray, Pa.), comprising a slit 42 through which a pipette tip (notshown) can be inserted to recover all or a portion of the concentratedliquid sample 24. In some embodiments (not shown), the sample port maycomprise a valve that can be opened to release a portion (or all) of theconcentrated liquid sample by gravity flow, for example.

FIG. 13 shows another embodiment of a device 10 according to the presentdisclosure. In this embodiment, material used for the filter membrane 14is configured to form a pocket 18 (e.g., by sealing together all but aportion of the edges of two sheets of filter membrane material) disposedinside a container 12 (e.g., a ZIPLOK bag). The pocket 18 has ininterior volume defined by the filter membrane 14. Optionally, thepocket 18 may further comprise an exterior nonwoven backing 20 toprovide structural support for the filter membrane 14. An absorbentmember 16 is placed in a portion of container 12 outside of the pocket18 formed by the filter membrane 14. Preferably, the absorbent member 16is proximate the pocket 18. More preferably, the absorbent member 16contacts at least a portion of the filter membrane 14 external to thepocket 18. Even more preferably, the absorbent member 16 is disposed onat least a portion of the filter membrane 14 external to the pocket 18.In some embodiments, the absorbent member 16 is disposed between thefilter membrane and the nonwoven backing 20. Optionally, the device 10further can comprise a support member 28 (e.g., a molded plastic rodwith a base) that can be used to provide structural support for arelatively flexible container 12 before, during, and after the additionof a liquid sample. In use, the device 10 can be held (e.g., eithermanually or via a support member) while a liquid sample (not shown) isplaced into the pocket 18. After a portion of the liquid sample passesthrough the filter membrane 14, the concentrated liquid sample (notshown) can be removed from the pocket 18 using a pipette, for example.

An alternative embodiment (not shown) of the device illustrated in FIG.13 comprises a device in which the absorbent member is disposed in theinterior volume of the pocket and the sample is deposited into thecontainer external to the pocket. In use, the sample is deposited intothe container rather than into the pocket. After at least a portion ofthe liquid sample has passed through the filter membrane, all or aportion of the remainder of the liquid sample in the container can bewithdrawn from the container for detection of a biological organismusing any of the detection methods disclosed herein.

The absorbent member 16 may be constructed of any suitablefluid-absorbing material. In some cases, the absorbent member 24 caninclude a hydrogel. As used herein, the term “hydrogel” refers to apolymeric material that is hydrophilic and that is either swollen orcapable of being swollen with a polar solvent. Suitable hydrogelsinclude crosslinked hydrogels, swollen hydrogels, and dried orpartially-dried hydrogels.

Suitable hydrogels include polymers comprising ethylenically unsaturatedcarboxyl-containing monomers and co-monomers selected from carboxylicacids, vinyl sulfonic acid, cellulosic monomer, polyvinyl alcohol, asdescribed in U.S. Patent Application Publication No. US2004/0157971;polymers comprising starch, cellulose, polyvinyl alcohol, polyethyleneoxide, polypropylene glycol, and copolymers thereof, as described inU.S. Patent Application Publication No. US 2006/0062854; the hydrogels,and polymeric beads made therefrom, described in International PatentPublication No. WO 2007/146722; polymers comprising polyurethaneprepolymer with at least one alcohol selected from polyethylene glycol,polypropylene glycol, and propylene glycol, as described in U.S. Pat.No. 6,861,067; and polymers comprising a hydrophilic polymer selectedfrom polysaccharide, polyvinylpyrolidone, polyvinyl alcohol, polyvinylether, polyurethane, polyacrylate, polyacrylamide, collagen and gelatin,as described in U.S. Pat. No. 6,669,981, the disclosures of which areall herein incorporated by reference in their entirety. Other suitablehydrogels include agar, agarose, and polyacrylamide hydrogels. Incertain embodiments, the hydrogel can include crosslinked polyacrylicacid sodium salt/copolymer, polyacrylamide copolymer, ethylene maleicanhydride copolymer, cross-linked carboxy-methyl-cellulose, polyvinylalcohol copolymers, cross-linked polyethylene oxide, starch graftedcopolymer of polyacrylonitrile, or any combination thereof.

The hydrogels can include a shaped hydrogel. Shaped hydrogels includehydrogels shaped into, for example, beads, sheets, ribbons, and fibers.

The hydrogels can further include supported hydrogels. Supportedhydrogels include hydrogels disposed on and/or in beads, nonwovensheets, fibers (e.g., blown microfibers), and the like.

In some embodiments, the volume of the concentrated sample may be lessthan 50% of the volume of the sample added to the device, although themethod may be practiced while reducing the volume of the sample to alesser extent. Exemplary degrees of volume reduction can include, forexample, the concentrated sample having a volume that is less than 40%,less than 35%, less than 30%, less than 25%, less than 20%, less than15%, less than 10%, less than 9%, less than 8%, less than 7%, less than6%, less than 5%, less than 4%, less than 3%, less than 2%, less than1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, lessthan 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than0.02%, or less than 0.01% of the volume of the sample added to thedevice. In one exemplary embodiment, an initial sample of 225 ml may bereduced to a volume of approximately 2 ml, so that the concentratedsample possesses a volume of approximately 0.09% of the volume of theliquid sample added to the device.

The methods further include detecting at least one biological organismretained by the filter membrane. In this context, a biological organismretained by the filter membrane includes biological organisms that arein contact with the filter membrane as well as biological organismssubsequently recovered from the filter membrane. Biological organismsmay be recovered from the filter membrane by any suitable method. Onefeature of biological organisms retained on the filter membrane bypracticing the methods described herein is that the retained biologicalorganisms may be removed from the filter membrane using relatively lowstringency physical methods such as, for example, gravity, manualshaking, and/or vortexing. So, for example, in embodiments justdescribed in which the volume of liquid sample is reduced, biologicalorganisms concentrated in the reduced volume of the liquid sample may beconsidered recovered from the filter membrane, even if the biologicalorganisms did not reside in or on the filter membrane for anydiscernable length of time.

Thus, in some embodiments, retained biological organisms may be detectedin situ while still in contact with the filter membrane. In otherembodiments, however, the retained biological organisms may be removedfrom the filter membrane and the biological organisms so recovered maybe detected. Whether detected in situ or following recovery from thefilter membrane, the biological organisms may be detected using anysuitable method including those routine to those of ordinary skill inthe art of microbial detection. Suitable in situ detection methodsinclude, for example, detecting biological organism-specific binding ofan antibody composition (e.g., monoclonal or polyclonal antibodies).Other detection methods can include, for example, detecting the presenceof a biological analyte produced by the biological organism. Exemplarydetection methods include, but are not limited to, detecting amplified(by, for example, PCR) biological organism-specific nucleotidessequences, nucleotide sequencing, enzyme assays (e.g., detection ofdehydrogenases, glucoronidases, β-galactosidases, proteases, etc.),bioluminescence assays (e.g., detection of ATP/ADP/AMP), detection ofproteins/peptides, spectrometry, and/or fluorescence (e.g., detection ofNAD/NADH, FAD/FADH, autofluorescence), and the like. Suitable detectionmethods for biological organisms recovered from the filter membraneinclude methods applicable for in situ detection of biologicalorganisms, and further includes detecting growth in culture.

In some cases, the method can further include quantifying biologicalorganisms retained by the filter membrane. In this context, too, abiological organism retained by the filter membrane includes biologicalorganisms that are in contact with the filter membrane as well asbiological organisms subsequently recovered from the filter membrane.

Thus, in some embodiments, retained biological organisms may bequantified in situ while still in contact with the filter membrane. Inother embodiments, however, the retained biological organisms may beremoved from the filter membrane and the biological organisms sorecovered may be quantified. Whether quantified in situ or followingrecovery from the filter membrane, the biological organisms may bequantified using any suitable method including those routine to those ofordinary skill in the art of microbial detection such as, for example,colony forming unit (cfu) detection, most probable number (MPN)analysis, ATP bioluminescence, enzyme assays, PCR, reverse transcriptasePCR (RT-PCR), quantitative PCR, and the like.

In embodiments in which the biological organisms are detected and/orquantified following recovery from the filter membrane, the methodincludes eluting at least 50% of the retained biological organisms fromthe filter membrane, although the method may be performed after elutingless than 50% of the retained biological organisms from the filtermembrane. In exemplary methods, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 76%, at least77%, at least 78%, at least 79%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, at least 97.5%, at least 98.5%, atleast 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%,or at least 99.9% of the retained biological organisms are eluted fromthe filter membrane.

In some cases, the retained biological organisms are eluted byrepositioning the filter membrane so that the force of gravity causesthe retained biological organisms to dislodge and thereby elute from thefilter membrane. In other cases, retained biological organisms may beeluted from the filter membrane by manually shaking the filter membraneto dislodge the retained biological organisms from the filter membrane.In other cases, retained biological organisms may be eluted by vortexingthe filter membrane to dislodge the retained biological organisms fromthe filter membrane. In other cases, biological organisms may be elutedfrom the filter membrane by foam elution as described in Example 12,below.

Certain existing methods provide recovery of up to about 30% ofbiological organisms (e.g., bacteria) and, therefore, fail to providethe same degree of recovery as observed using the methods describedherein.

Without wishing to be bound by any particular theory, certain existingmethods may fail to provide satisfactory recovery of biologicalorganisms because the microporous filter membranes possess a significantamount of large pores at the surface of the membrane even when thefilter membranes have a pore rating smaller than the size of bacteria.The large pores are believed to entrap the biological organisms ratherthan retain the biological organisms while the sample volume is beingreduced.

In addition, nonspecific binding of, for example, bacteria to thesurface of the filter membrane creates a challenge. This is due, atleast in part, because one way to reduce the volume of a liquid samplemore quickly is to provide a greater surface area of filter membranethrough which liquid may be absorbed. Increasing the surface are,however, also increases the surface area to which biological organismsmay bind nonspecifically. Many alternatives were investigated. However,none of the membranes was able to provide both a high retention rate ofbiological organisms (and, therefore, high recovery rate) and a goodwater flux volume (e.g., sufficient to concentrate from 225 ml to 2-3 mlin, for example, less than one hour.

In certain embodiments, the method can provide a reduction in samplevolume of about 98.6% to about 99.2% while permitting recovery of atleast 70% of biological organisms in the original sample.

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1 Preparation of TIPS Membranes R1901-11 Membrane

A multi-zone microporous polypropylene membrane (designated herein asR1901-11) was prepared as described in International Patent PublicationNo. WO2010/078234 using both a 40 mm twin screw extruder and a 25 mmtwin screw extruder. Melt streams from the two extruders were cast intoa single sheet through a multi-manifold die.

Melt stream 1. Polypropylene (PP) resin pellets (F008F from SunocoChemicals, Philadelphia, Pa.) and a nucleating agent (MILLAD 3988,Milliken Chemical, Spartanburg, S.C.) were introduced into a 40 mm twinscrew extruder which was maintained at a screw speed of 250 rpm. Themineral oil diluent (Mineral Oil SUPERLA White 31, Chevron Corp., SanRamon, Calif.) was fed separately from the reservoir into the extruder.The weight ratio of PP/diluent/nucleating agent was 29.25%/70.7%/0.05%.The total extrusion rate was about 30 lb/hr (13.6 kg/hr) and theextruder's eight zones were set to provide a decreasing temperatureprofile from 271° C. to 177° C.

Melt stream 2. PP resin pellets and MILLAD 3988 were introduced into a25 mm twin screw extruder which was maintained at a screw speed of 125rpm. The mineral oil diluent was fed separately from the reservoir intothe extruder. The weight ratio of PP/diluent/nucleating agent was29.14%/70.7%/0.16%. The total extrusion rate was about 6 lb/hr (2.72kg/hr) and the extruder's eight zones were set to provide a decreasingtemperature profile from 271° C. to 177° C.

The multi-zone film was cast from the multi-manifold die maintained at177° C. onto a patterned casting wheel. The temperature of casting wheelwas maintained at 60° C. and the casting speed was 3.35 m/min (11ft/min) The resulting film was washed in-line in a solvent to removemineral oil in the film and then air dried. The washed film wassequentially oriented in the length and cross direction 1.8×2.80 at 99°C. and 154° C., respectively.

R1901-8B Membrane

A multi-zone microporous polypropylene membrane (designated herein asR1901-8B) was prepared as described in International Patent PublicationNo. WO2010/078234 using both a 40 mm twin screw extruder and a 25 mmtwin screw extruder. Melt streams from the two extruders were cast intoa single sheet through a multi-manifold die.

Melt stream 1. Polypropylene (PP) resin pellets (F008F from SunocoChemicals, Philadelphia, Pa.) and a nucleating agent (MILLAD 3988,Milliken Chemical, Spartanburg, S.C.) were introduced into a 40 mm twinscrew extruder which was maintained at a screw speed of 250 rpm. Themineral oil diluent (Mineral Oil SUPERLA White 31, Chevron Corp., SanRamon, Calif.) was fed separately from the reservoir into the extruder.The weight ratio of PP/diluent/nucleating agent was29.254%/70.7%/0.045%. The total extrusion rate was about 27 lb/hr (12.2kg/hr) and the extruder's eight zones were set to provide a decreasingtemperature profile from 271° C. to 177° C.

Melt stream 2. PP resin pellets and MILLAD 3988 were introduced into a25 mm twin screw extruder which was maintained at a screw speed of 125rpm. The mineral oil diluent was fed separately from the reservoir intothe extruder. The weight ratio of PP/diluent/nucleating agent was28.146%/70.7%/0.154%. The total extrusion rate was about 9 lb/hr (4.08kg/hr) and the extruder's eight zones were set to provide a decreasingtemperature profile from 271° C. to 177° C.

The multi-zone film was cast from the multi-manifold die maintained at177° C. onto a patterned casting wheel. The temperature of casting wheelwas maintained at 60° C. and the casting speed was 3.52 m/min (11.54ft/min). The resulting film was washed in-line in a solvent to removethe mineral oil diluent and then air dried. The washed film wassequentially oriented in the length and cross direction 1.6×2.85 at 99°C. and 154° C., respectively.

R1933-7 Membrane

A multi-zone microporous polypropylene membrane (designated herein asR1933-7) was prepared as described in International Patent PublicationNo. WO2010/078234 using both a 40 mm twin screw extruder and a 25 mmtwin screw extruder. Two melt streams from extruders were cast into asingle sheet through a multi-manifold die.

Melt stream 1. Polypropylene (PP) resin pellets (F008F from SunocoChemicals, Philadelphia, Pa.) and a nucleating agent (MILLAD 3988,Milliken Chemical, Spartanburg, S.C.) were introduced into a 40 mm twinscrew extruder which was maintained at a screw speed of 175 rpm. Themineral oil diluent (Kaydol 350 Mineral Oil, Brenntag Great Lakes LCC,St. Paul, Minn.) was fed separately from a reservoir into the extruder.The weight ratio of PP/diluent/nucleating agent was34.247%/65.7%/0.053%. The total extrusion rate was about 32 lb/hr (14.5kg/hr) and the extruder's eight zones were set to provide a decreasingtemperature profile from 271° C. to 177° C.

Melt stream 2. PP resin pellets and MILLAD 3988 were introduced into a25 mm twin screw extruder which was maintained at a screw speed of 150rpm. The mineral oil diluent was fed separately from the reservoir intothe extruder. The weight ratio of PP/diluent/nucleating agent was29.14%/70.7%/0.16%. The total extrusion rate was about 6 lb/hr (2.72kg/hr) and the extruder's eight zones were set to provide a decreasingtemperature profile from 254° C. to 177° C.

The multi-zone film was cast from the multi-manifold die maintained at177° C. onto a patterned casting wheel. The temperature of casting wheelwas maintained at 71° C. and the casting speed was 5.79 m/min (19.00ft/min). The resulting film was washed in-line in a solvent to removemineral oil diluent and then air dried. The washed film was sequentiallyoriented in the length and cross direction 1.5×2.70 at 99° C. and 160°C., respectively.

R1933-18 Membrane

A multi-zone microporous polypropylene membrane (designated herein asR1933-18) was prepared as described in International Patent PublicationNo. WO2010/078234 using both a 40 mm twin screw extruder and a 25 mmtwin screw extruder. Two melt streams from extruders were cast into asingle sheet through a multi-manifold die.

Melt stream 1. Polypropylene (PP) resin pellets (F008F from SunocoChemicals, Philadelphia, Pa.) and a nucleating agent (MILLAD 3988,Milliken Chemical, Spartanburg, S.C.) were introduced into the hopperusing a solids feeder and the materials were fed into of a 40 mm twinscrew extruder which was maintained at a screw speed of 175 rpm. Themineral oil diluent (Kaydol 350 Mineral Oil, Brenntag Great Lakes LCC,St. Paul, Minn.) was fed separately from a reservoir into the extruder.The weight ratio of PP/diluent/nucleating agent was34.247%/65.7%/0.053%. The total extrusion rate was about 32 lb/hr (14.5kg/hr) and the extruder's eight zones were set to provide a decreasingtemperature profile from 271° C. to 177° C.

Melt stream 2. PP resin pellets and MILLAD 3988 were introduced into a25 mm twin screw extruder which was maintained at a screw speed of 150rpm. The mineral oil diluent was fed separately from the reservoir intothe extruder. The weight ratio of PP/diluent/nucleating agent was28.98%/70.7%/0.32%. The total extrusion rate was about 6 lb/hr (2.72kg/hr) and the extruder's eight zones were set to provide a decreasingtemperature profile from 260° C. to 194° C.

The multi-zone film was cast from the multi-manifold die maintained at177° C. onto a patterned casting wheel. The temperature of casting wheelwas maintained at 52° C. and the casting speed was 5.84 m/min (19.15ft/min). The resulting film was washed in-line in a solvent to removethe mineral oil diluent and then air dried. The washed film wassequentially oriented in the length and cross direction 1.7×2.75 at 99°C. and 160° C., respectively.

Example 2 Surface Coating of TIPS Membranes

A 4-wt % SPAN20 (Uniqema, New Castle, Del.) solution was prepared bydissolving the surfactant in 2-propanol (Alfa Aesar, Ward Hill, Mass.).

A TIPS microporous membrane was saturated with the above surfactantsolution in a polyethylene (PE) bag. The membrane saturated instantlyand excessive surface solution was removed by rubbing the PE bag. Themembrane was removed from the bag and exposed to air to completely drythe membrane. The dried membranes were stored in a PE bag at roomtemperature.

Example 3 Surface Modification of TIPS Membranes

The TIPS membranes were coated with polyethylene glycol (PEG) asdescribed in U.S. Provisional Patent Application Ser. No. 61/351,447,entitled, “Process for Making Coated Porous Materials,” filed Jun. 4,2010.

A 5-wt % EVAL stock solution was made by dissolving an ethylene-vinylalcohol copolymer (EVAL) with 44 mol % ethylene content (EVAL44,Sigma-Aldrich Co., St Louis, Mo., USA) in an ethanol (AAPER Alcohol andChemical Co. Shelbyville, Ky.)/water solvent mixture (70 vol % ethanol)in a water bath at temperature 70-80° C.

From the above stock solution, a solution was made containing 1-wt %EVAL44, 2-wt % SR@610 (Sartomer, Warrington, Pa.), 1 wt % reactivephotoinitiator VAZPIA(2-[4-(2-hydroxy-2-methylpropanoyl)phenoxy]ethyl-2-methyl-2-N-propenoylaminopropanoate, as disclosed in U.S. Pat. No. 5,506,279) in ethanol/watermixture solvent (70 vol % ethanol)

A TIPS microporous membrane was saturated with the coating solutionabove in a heavy weight PE bag. Effort was made to remove the excessivesurface solution by paper towel wiping after the saturated membrane wasremoved from the PE bag. The membrane was allowed to dry by solventevaporation at room temperature for 10-12 hours. Then, the dry membranewas saturated with a 20-wt % NaCl aqueous solution. After that, themembrane went through a nitrogen inert Fusion UV system with H-bulb on aconveying belt. The speed of the belt was 20 feet per minute (fpm). Themembrane was sent through the UV system again in the same speed with theopposite membrane side facing the light source. The cured membranesample was washed in excessive deionized water and dried at 90° C. for 1to 2 hours until completely dry. The dried membranes were stored in a PEbag at room temperature.

Example 4 Preparation of Polyacrylonitrile (PAN) Membranes

Various PAN membranes were made as disclosed in Korean PatentApplication No. KR20040040692. A 10.5-wt % of polyacrylonitrile (Mw.150,000) was prepared in N,N-dimethlyacetamide (DMAC). Using a syringepump, a constant flow of PAN polymer solution (50 μl/min/hole) wassupplied into a syringe connected to a high voltage source. An electricforce of 90-100 Kv was introduced to form an electrostatic force tocause the polymer solution ejection into air and formation of PANnanofibers. After the electrospinning process, the collected PANnanofibers had bulkiness similar to cotton and not like that of a filmand/or a membrane. To reduce the bulkiness and to increase thestructural integrity of the electrospun PAN nanofibers, a post-treatment(hot calendaring process) was carried out at 140° C. and 10-20 kgf/cm³pressure. The PAN nanofibers were stored as a roll in a PE bag at roomtemperature.

Example 5 Characterization of Membranes a) Water Flow Rate Measurement

A 47 mm disk of a membrane was cut using a die punch and the membranedisk was mounted in a Gelman magnetic holder (Gelman Sciences, Inc., AnnArbor, Mich.). The active membrane diameter in the holder was 34 mm. Onehundred ml of water was added to the holder and a vacuum pressure ofabout 23.5 inches of mercury was applied using a vacuum pump (GASTManufacturing, Inc., Benton Harbor, Mich.) to draw water through themembrane. The time for the water to pass through the membrane wasrecorded with a stopwatch. The water flow rate (flux) was calculatedusing the time, vacuum pressure, and area of the membrane and expressedin L/(m²·h·psi).

b) Bubble Point Pore Size Measurement

The Bubble Point pore size of a membrane was measured according toASTM-F316-03. The membrane was pre-wetted with isopropanol or FC-43 (3MCo., St Paul, Minn.), or liquid GALWICK (PMI, Porous Materials, Inc.,Ithaca, N.Y.) and mounted on a testing holder. Pressurized nitrogen gaswas gradually applied to one side of the membrane until the gas flowdetected at the other side reached 100%. The pressure at 100% gas flowthrough the membrane was recorded and used to calculated Bubble Pointpore size.

TIPS membrane processing conditions are summarized in Table 1, below.

The water flux rate and Bubble Point pore size for various membranes areshown below in Table 2, below.

TABLE 1 TIPS Membrane Processing Conditions R1930-10 R1901-11 R1901-8BR1933-7 R1933-18 MS 1 Screw Speed 150 rpm 250 rpm 250 rpm 175 rpm 175rpm MS 1 PP/ 29.23/70.70/0.072 29.25/70.7/0.05 29.25/70.7/0.04534.25/65.7/0.053 34.25/65.7/0.053 DIL/ NA ratio (weight %) MS 1Extrusion 21 lbs/hr 30 lb/hr 27 lb/hr 32 lb/hr 32 lb/hr Rate (9.53 kg/h)(13.6 kg/hr) (12.2 kg/hr) (14.5 kg/hr) (14.5 kg/hr) MS 1 Temp Profile271° C. to 204° C. 271° C. to 177° C. 271° C. to 177° C. 271° C. to 177°C. 271° C. to 177° C. MS 2 Screw Speed 150 rpm 125 rpm 125 rpm 150 rpm150 rpm MS 2 PP/ 29.15/70.70/0.15 29.14/70.7/0.16 29.15/70.7/0.1529.14/70.7/0.16 28.98/70.7/0.32 DIL/ NA ratio (weight %) MS 2 Extrusion 9 lbs/hr  6 lb/hr  9 lb/hr  6 lb/hr  6 lb/hr Rate (4.08 kg/h) (2.72kg/hr) (4.08 kg/hr) (2.72 kg/hr) (2.72 kg/hr) MS 2 Temp Profile 271° C.to 204° C. 271° C. to 177° C. 271° C. to 177° C. 254° C. to 177° C. 260°C. to 194° C. Die Temperature 199° C. (390° F.) 177° C. (350° F.) 177°C. (350° F.) 177° C. (350° F.) 177° C. (350° F.) Wheel temperature  60°C.  60° C.  60° C.  71° C.  52° C. Casting wheel 13.0 ft/min 3.35 m/min3.52 m/min 5.79 m/min 5.84 m/min speed (4.0 m/min) (11 ft/min) (11.54ft/min) (19.00 ft/min) (19.15 ft/min) Orientation - 1.70 × 3.35 1.8 ×2.80 1.6 × 2.85 1.5 × 2.70 1.7 × 2.75 L × W Orientation Temp-  99°C./154° C.  99° C./154° C.  99° C./54° C.  99° C./160° C. L/W

TABLE 2 Membrane Properties TIPS Membrane Water Bubble Tight flux Pointzone Total (L/m² · pore size Por- thickness thickness Membrane -treatment h · psi) (μm) osity (μm) (μm) R1930-10 - untreated 937 0.3484% 16.0 53.3 R1930-10 - SPAN20 917 — 16.0 53.3 R1901-11 - untreated2723 0.74 85% 8 104 R1901-11 - SPAN20 2,739 0.74 — 8 104 R1901-11 - PEG2427 0.62 — 8 104 R1902-8B - untreated 1832 0.51 84% 23 109 R1902-8B -SPAN20 1,945 0.51 84% 23 109 R1902-8B - PEG 2091 0.49 — 23 109 R1933-7 -untreated 1263 0.34 77% 12 74 R1933-7 - SPAN20 680 0.34 — 12 74R1933-7 - PEG 1401 0.34 — 12 74 R1933-18 - untreated 577 0.23 — 6 56R1922-18 - SPAN20 357 — — 6 56 Nanofiber filters PAN-1 3995 0.613 62% —11.1 PAN-2 2430 0.531 62% — 16.9 PAN-3 3436 0.367 70% — 15.5

c) Scanning Electron Microscopy of Membranes

For PAN membranes, the filter from each of the samples was mounted on analuminum stub. For TIPS membranes, two sections from each of the sampleswere removed and mounted on an aluminum stub to view both the “Tight”and “Open” surfaces. Cross sections of each of the TIPS membranes werealso prepared by tearing under liquid nitrogen. These were mounted on anadditional stub. All specimens were sputter coated with gold/palladiumand were examined using a JEOL 7001F Field Emission Scanning ElectronMicroscope. Digital photomicrographs were the product of secondaryelectron imaging (SEI), a technique used to image surface morphology ofa sample. All micrographs were taken at a viewing angle normal to thesurface of the stub or sectioned face (nominally). Images were capturedat various magnifications and the magnification is indicated on theimages shown. The “Tight” and “Open” surfaces (also referred to as sidesor zones) are indicated in the image for each cross section. A lengthmarker is also shown in the lower portion of each micrograph of FIGS.1-6.

Example 6 Bacteria Used in Examples

The various bacteria used in the examples (Table 3) were obtained fromATCC (Manassas, Va.).

TABLE 3 Bacteria used in examples Bacteria ATCC No. Enterococcusfaecalis 700802  Escherichia coli 51813 Salmonella enterica subsp.enterica 51812 Citrobacter braakii 10625 Citrobacter freundii 14135Enterobacter aerogenes 29007 Enterobacter cloacae 10699

Pure cultures of the bacterial strains were inoculated into Tryptic SoyBroth (TSB, BD, Franklin Lakes, N.J.) and were grown overnight at 37° C.The cultures were diluted serially in Butterfield phosphate buffer(Whatman, Piscataway, N.J.) to obtain desired amount of colony formingunits (cfu) per ml for spiking into water samples. The bacteria werequantified by plating appropriate dilutions on 3M PETRIFILM E.coli/Coliform Count Plates (3M Co., St. Paul, Minn.) according tomanufacturer's instruction and incubated overnight at 37° C. The plateswere read using 3M PETRIFILM Plate Reader (3M Co.) and colony formingunits (cfu) were determined.

Example 7 Recovery of E. coli from Spiked Water Samples by FiltrationFollowed by Direct Growth

E. coli was grown over night in Tryptic Soy Broth (TSB) at 37° C. Theculture was diluted to obtain approximately 100 cfu/ml and 1 ml of thesolution was added to 1000 ml of sterile water to obtain approximately100 cfu. A 47 mm membrane was cut from sheets or discs and placed onsterile glass filter holder assembly with funnel, flitted base(Millipore, Billerica, Mass.). The filter holder was connected to a 4 Lvacuum filtering flask. The solution was filtered through the variousmembranes at vacuum pressure of about 20 inches of mercury using an AIRCADET Vacuum/Pressure Station (model No. 420-3901, Barnant Company,Barrington, Ill.). The membranes were removed aseptically and placed onblood agar or tryptic soy agar plates (Hardy Diagnostics, Santa Maria,Calif.) and incubated overnight at 37° C. The colonies growing onmembranes were counted to determine colony forming units (cfu). Theresults obtained are shown below in Table 4. All the membranes testedshowed greater than 73% recovery with the smaller pore size membranes(<0.5 μm) showing greater than 90% recovery.

TABLE 4 Recovery of E. coli from spiked water sample by filtration anddirect growth Total cfu recovered % Recovery Input 105 cfu R1933-18/SPAN(0.23 μm) 98 93.33 R1933-7/SPAN (0.34 μm) 95 90.48 R1901-8B/SPAN (0.49μm) 92 87.62 R1901-11/SPAN (0.74 μm) 77 73.33 PAN-1 (0.613 μm) 80 76.19PAN-2 (0.531 μm) 88 83.81 PAN-3 (0.367 μm) 100 95.24 Isoporepolycarbonate filter (0.40 μm) 97 92.38 MF-Millipore Type HAWP (0.45 μm)98 93.33

Example 8 Recovery of E. coli from Spiked Water Samples by FiltrationFollowed by Elution

E. coli was grown over night in TSB at 37° C. The culture was diluted toobtain approximately 100 cfu/ml and 1 ml of the solution was added to1000 ml of sterile water to obtain approximately 100 cfu. A 47 mmmembrane was cut from sheets or discs and placed on sterile vacuumfiltration apparatus. The solution was filtered through the variousmembranes at vacuum pressure of about 20 inches of mercury using an AIRCADET Vacuum/Pressure Station. The membranes were removed asepticallyand added to a sterile polystyrene 50-ml centrifuge tube (BDBiosciences, San Jose, Calif.) with 5 ml of 0.2% Tween-20 (Sigma-AldrichCo., St. Louis, Mo.) and vortexed (Fixed Speed Vortex Mixer, VWR, WestChester, Pa.) at room temperature for 1 to 2 minutes. The solution wasplated on 3M PETRIFILM E. coli/Coliform Count Plates (3M Co., St. Paul,Minn.) according to manufacturer's instruction and incubated overnightat 37° C. The plates were read using 3M PETRIFILM Plate Reader (3M Co.)and colony forming units (cfu) were determined The results obtained areshown below in Table 5. The results are representative of a typicalexperiment. From 1000 ml water samples spiked with about 100 cfu of E.coli, recoveries ranged from 28.6% to 72.9%. For the treated TIPSmembranes and one PAN membrane (PAN-3) the recovery varied from 45% to72.9%. The TIPS membrane R1933-18/SPAN (0.2 μm) had a low flux rate asit took 25 minutes to filter 1 liter of water. R1933-7/SPAN (0.34 μm)had a good flux rate. Both membranes showed good recovery of filteredbacteria. For the two commercial membranes the recovery ranged from 28.6to 35.7%.

TABLE5 Recovery of E. coli by filtration and elution from variousmembranes Time to filter 1 liter 20 mm Total cfu % Hg vacuum recoveredRecovery (min:sec) Input 140 cfu R1933-18/SPAN (0.2 μm) 102 72.86 25:19 R1933-7/Original (0.34 μm) 54 38.57 5:28 R1933-7/PEG (0.34 μm) 87 61.906:01 R1933-7/SPAN (0.34 μm) 98 70.00 6:08 R1901-8B/PEG (0.51 μm) 6848.81 2:50 R1901-8B/SPAN (0.49 μm) 78 55.71 2:56 R1901-11/PEG (0.62 μm)63 45.24 2:11 R1901-11/SPAN (0.74 μm) 72 51.43 2:40 PAN-1 (0.613 μm) 4532.14 1:08 PAN-2 (0.531 μm) 52 37.14 1:32 PAN-3 (0.367 μm) 100 71.431:20 Isopore Polycarbonate filter (0.40 50 35.71 2:41 μm) MF-MilliporeType HAWP 40 28.57 2:35 (0.45 μm)

Example 9 Effect of Various Extractants on Recovery of Bacteria fromSpiked Water Samples by Filtration Followed by Elution

E. coli was grown over night in TSB at 37° C. The culture was diluted toobtain approximately 100 cfu/ml and 1 ml of the solution was added to1000 ml of sterile water to obtain approximately 100 cfu. A 47 mmmembrane was cut from sheets or discs and placed on a vacuum filtrationapparatus. The solution was filtered through the various membranes atvacuum pressure of about 20 inches of mercury using an AIR CADETVacuum/Pressure Station. The membranes were removed aseptically andadded to a sterile polystyrene 50-ml centrifuge tube (BD Biosciences,San Jose, Calif.) with 5 ml of various extractants and vortexed (FixedSpeed Vortex Mixer, VWR, West Chester, Pa.) at room temperature for 1 to2 minutes. The solution was plated on 3M PETRIFILM E. coli/ColiformCount Plates according to manufacturer's instruction and incubatedovernight at 37° C. The plates were read using 3M PETRIFILM Plate Readerand colony forming units (cfu) were determined. The results obtained areshown below in Table 6. Use of 0.2% Tween-20 and phosphate bufferedsaline (PBS, Invitrogen, Carlsbad, Calif.) showed better recovery thanTriton-X-100 (Sigma-Aldrich Co., St. Louis, Mo.) or water. WithTween-20, the recovery ranged from 35% to 77%, while with PBS it was 33%to 79%. With sterile MILLI-Q water (Millipore Corp., Billerica, Mass.)and Trition-X-100 the recoveries were only 11% to 46%.

TABLE 6 Recovery of E. coli by filtration and elution using variousextractants from membranes 0.2% 0.1% Triton- Milli-Q Membranes Tween-20X-100 PBS Water R1933-7/SPAN (0.34 μm) 76.7% 37.1% 73.8% 26.2%R1901-8B/SPAN (0.49 μm) 65.2% 31.4% 69.0% 35.7% PAN-3 (0.367 μm) 63.5%46.2% 78.6% 35.7% Isopore Polycarbonate 34.7% 10.7% 33.3% 19.0% (0.40μm)

Example 10

Effect of Tween-20 Concentrations on Recovery of Bacteria

E. coli was grown over night in TSB at 37° C. The culture was diluted toobtain approximately 100 cfu/ml and 1 ml of the solution was added to1000 ml of sterile water to obtain approximately 100 cfu. A 47 mmmembrane was cut from sheets or discs and placed on a vacuum filtrationapparatus. The solution was filtered through the various membranes atvacuum pressure of about 20 inches of mercury using an AIR CADETVacuum/Pressure Station. The membranes were removed aseptically andadded to a sterile polystyrene 50-ml centrifuge tube (BD Biosciences,San Jose, Calif.) with 5 ml of various concentrations of Tween-20 andvortexed (Fixed Speed Vortex Mixer, VWR, West Chester, Pa.) at roomtemperature for 1 to 2 minutes. The solution was plated on 3M PETRIFILME. coli/Coliform Count Plates according to manufacturer's instructionand incubated overnight at 37° C. The plates were read using 3MPETRIFILM Plate Reader and colony forming units (cfu) were determined.The results obtained are shown below in Table 7. Use of 0.1% or 0.2%Tween-20 gave the best recoveries from all of the membranes tested.

TABLE 7 Effect of Tween-20 concentration on percent Recovery of E. coliby filtration and elution from membranes Tween-20 concentrationMembranes 0.01% 0.05% 0.10% 0.20% 0.50% R1933-7/PEG (0.34 μm) 57.5 45.751.6 70.8 20.6 R1933-7/SPAN (0.34 μm) 64.9 57.5 59.0 78.2 73.7R1901-8B/PEG (0.51 μm) 38.3 47.2 63.4 67.8 23.6 PAN-3 (0.367 μm) 38.544.9 57.7 64.1 32.1 Isopore Polycarbonate filter 12.8 19.2 32.1 38.519.2 (0.40 μm) MF-Millipore Type HAWP 19.2 32.1 38.5 32.1 25.6 (0.45 μm)

Example 11 Effect of Various Methods for Recovery of Bacteria fromMembranes

E. coli was grown over night in TSB at 37° C. The culture was diluted toobtain approximately 100 cfu/ml and 1 ml of the solution was added to1000 ml of sterile water to obtain approximately 100 cfu. A 47 mmmembrane was cut from sheets or discs and placed on a vacuum filtrationapparatus. The solution was filtered through the various membranes atvacuum pressure of about 20 inches of mercury using an AIR CADETVacuum/Pressure Station. The membranes were removed aseptically andadded to a sterile polystyrene 50-ml centrifuge tube (BD Biosciences,San Jose, Calif.) with 5 ml of 0.2% Tween-20. The tubes were sonicatedfor 5 minutes using an ultrasonicator (Branson 2200, BransonUltrasonics, Dansbury, Conn.), vortexed for 1 to 2 minutes (Fixed SpeedVortex Mixer, VWR, West Chester, Pa.) or shaken in an orbital shaker(Newbrunswik Scientific shaker, Model Innova 4000) for 10 minutes atroom temperature. The solutions were plated on 3M PETRIFILM E.coli/Coliform Count Plates according to manufacturer's instruction andincubated overnight at 37° C. The plates were read using 3M PETRIFILMPlate Reader and colony forming units (cfu) were determined. The resultsobtained are shown below in Table 8.

TABLE 8 Recovery of E. coli from membranes after filtration by variousmethods of extraction Sonication Vortexing Shaking Total cfu % Total cfu% Total cfu % recovered recovery recovered recovery recovered recoveryInput cells 96 cfu R1933-7/PEG (0.34 μm) 28 29.5 63 66 12 12.5R1901-8B/PEG (0.51 μm) 30 31.3 75 78.1 15 15.6 PAN-3 (0.367 μm) 32 33.047 48.6 32 33.3 Isopore 22 22.6 42 43.4 15 15.6 polycarbonate filter(0.40 μm) MF-Millipore Type 28 29.5 38 39.9 10 10.4 HAWP (0.45 μm)

Example 12 Recovery of Bacteria from Membranes Using Foam Elution

E. coli was grown over night in TSB at 37° C. The culture was diluted toobtain approximately 100 cfu/ml and 1 ml of the solution was added to 50ml of sterile water to obtain approximately 100 cfu. A 25 mm membranewas cut from sheets or discs and placed in a 25 mm Swinnex filter holder(Millipore Corp., Billerica, Mass.). The filter holder was attached to avacuum manifold (Waters Corporation, Milford, Mass.) and a 50 ml syringewas attached to the other end of filter holder. The spiked water samplewas filtered through the various membranes at vacuum pressure of about20 inches of mercury using an AIR CADET Vacuum/Pressure Station. Thefilter holder with the membrane was attached to HSC 40 bench-topconcentrator (InnovaPrep, Drexel, Mo.). The system generates foam of theextractant solution and the bacteria were eluted by passing the foam (1ml of 0.05% Tween-20) through the membrane.

The extracted solutions were plated on 3M PETRIFILM E. coli/ColiformCount Plates according to manufacturer's instruction and incubatedovernight at 37° C. The plates were read using 3M PETRIFILM Plate Readerand colony forming units (cfu) were determined. The results obtained areshown below in Table 9. The foam elution method offers an advantage foreluting biological organisms in small volumes and enables easyextraction of nucleic acids without further concentration of elutedmaterial.

TABLE 9 Recovery of E. coli from membranes after filtration by foamelution Total cfu in % 1 ml Recovery Input 86 cfu R1933-7/SPAN (0.34 μm)46 53.5 R1933-7/PEG (0.34 μm) 49 57.0 PAN-3 (0.367 μm) 47 54.7 Isoporepolycarbonate filter (0.40 μm) 35 40.7 MF-Millipore Type HAWP (0.45 μm)25 29.1

Example 13 Recovery of Bacteria from Spiked Water Samples Followed byGrowth

E. coli, Salmonella enterica subsp. enterica, and Enterococcus faecaliswere grown over night in TSB at 37° C. The culture was diluted to obtainapproximately 10 cfu/ml and 1 ml of the solution was added to 1000 ml ofsterile water to obtain approximately 10 cfu. The solution was filteredthrough the various membranes at vacuum pressure of about 20 inches ofmercury using an AIR CADET Vacuum/Pressure Station. The membranes wereremoved aseptically and added to a sterile polystyrene 50-ml centrifugetube (BD Biosciences, San Jose, Calif.) with 10 ml of Terrific Broth(TB) or tryptic soy broth (TSB) and agitated at 300 rpm in a NewbrunswikScientific shaker, Model Innova 4000 for 2 hours at 37° C. Control tubeswere set up by spiking about 10 cfu (100 μl of 10² cfu/ml) into 10 ml TBand were grown similarly. At the end of two hours, growth media from thetubes were plated on 3M PETRIFILM E. coli/Coliform Count Plates (for E.coli) and Aerobic Count Plates (for S. enterica and Enterococcusfaecalis) and incubated overnight at 37° C. The plates were read using3M PETRIFILM Plate Reader and colony forming units (cfu) weredetermined. The input number of cells was used to calculate thefold-increase.

TABLE 10 Increase in cell number of E. coli after filtration and growthin TB for two hours Total cfu Fold- Total cfu Fold- in 5 ml increase in5 ml increase Input to 1 liter water 6 cfu 24 cfu Control (nofiltration) 65 10.83 250 10.42 R1901-11/SPAN (0.74 μm) 68 11.33 195 8.13PAN-3 (0.367 μm) 67 11.17 225 9.38 Isopore polycarbonate filter 43 7.17105 4.38 (0.40 μm)

TABLE 11 Increase in cell number of E. coli after filtration and growthin TB for two hours Total cfu in Fold- 10 ml increase Input to I literwater 11 cfu Control (no filtration) 150 13.64 PAN-1 (0.613 μm) 70 6.36PAN-2 (0.531 μm) 100 9.09 PAN-3 (0.367 μm) 160 14.55 R1901-8B/SPAN (0.49μm) 140 12.73 R1901-11/SPAN (0.74 μm) 110 10.00 Isopore polycarbonatefilter (0.40 μm) 60 5.45 MF-Millipore Type HAWP (0.45 μm) 40 3.64

TABLE 12 Increase in E. coli cell numbers after filtration and growth inTB for two hours Total cfu in Fold- 10 ml Increase Input 11 cfu Control150 13.6 R1933-18/SPAN (0.2 μm) 120 10.9 R1933-7/PEG (0.34 μm) 130 11.8R1933-7/SPAN (0.34 μm) 120 10.9 R1901-8B/PEG (0.51 μm) 110 10.0R1901-8B/SPAN (0.49 μm) 140 12.7 R1901-11/PEG (0.62 μm) 90 8.2R1901-11/SPAN (0.74 μm) 100 9.1 PAN-3 (0.367 μm) 107 9.7 IsoporePolycarbonate filter (0.40 μm) 70 6.4 MF-Millipore Type HAWP (0.45 μm)60 5.5

TABLE 13 Increase in E. coli cell numbers after filtration and growth inTSB for two hours Total Total cfu Fold- cfu in Fold- in 10 ml increase10 ml increase Input 11 cfu 44 cfu Control 150 13.6 540 12.3R1901-8B/SPAN (0.49 μm) 145 13.2 350 8.0 PAN-3 (0.367 um) 130 11.8 3107.0 Isopore Polycarbonate filter (0.40 μm) 85 7.7 230 5.2 MF-MilliporeType HAWP (0.45 μm) 70 6.4 250 5.7

TABLE 14 Increase in S. enterica cell numbers after filtration andgrowth in TSB for two hours Total cfu Fold- Total cfu Fold- in 10 mlincrease in 10 ml increase Input 9 cfu 36 cfu Control 40 4.4 150 4.2R1901-8B/SPAN (0.49 μm) 35 3.9 130 3.6 PAN-3 (0.367 um) 30 3.3 150 4.2Isopore Polycarbonate filter 22 2.4 90 2.5 (0.40 μm) MF-Millipore TypeHAWP 25 2.8 110 3.1 (0.45 μm)

TABLE 15 Increase in E. coli and Enterococcus faecalis cell numbersafter filtration and growth inTSB for two hours Enterococcus E. colifaecalis Total Total cfu in Fold- cfu in Fold- 10 ml increase 10 mlincrease Input 13 cfu 30 cfu Control 130 10.0 220 7.4 R1933-7/PEG (0.34μm) 110 8.5 160 5.2 R1933-7/SPAN (0.34 μm) 120 9.2 180 6.0 IsoporePolycarbonate filter (0.40 μm) 70 5.4 130 4.2 MF-Millipore Type HAWP(0.45 μm) 50 3.8 60 2.0

Example 14 Recovery of Coliform Bacteria from Spiked Water SamplesFollowed by Growth

E. coli, Enterobacter aerogenes, Enterobacter cloacae, Citrobacterfreundii, and Citrobacter braakii were grown over night in TSB at 37° C.The culture was diluted to obtain approximately 100 cfu/ml and 0.4 ml ofthe solution was added to 1000 ml of sterile water to obtainapproximately 40 cfu. The solution was filtered through the variousmembranes at vacuum pressure of about 20 inches of mercury using an AIRCADET Vacuum/Pressure Station. The membranes were removed asepticallyand added to a sterile polystyrene 50-ml centrifuge tube (BDBiosciences, San Jose, Calif.) with 10 ml of Terrific Broth (TB,Sigma-Aldrich Co., St. Louis, Mo.) or tryptic soy broth (TSB, BDBiosciences, San Jose, Calif.) and agitated at 300 rpm in a NewbrunswikScientific shaker, Model Innova 4000 for 2.5 or 3 hours at 37° C.Control tubes were set up by spiking about 40 cfu (400 μl of 100 cfu/ml)into 10 ml TB and were grown similarly. At the end of incubation period,growth media from the tubes were plated on 3M PETRIFILM E. coli/ColiformCount Plates and incubated overnight at 37° C. The plates were readusing 3M PETRIFILM Plate Reader and colony forming units (cfu) weredetermined. The input number of cells was used to calculate thefold-increase.

TABLE 16 Increase in cell number of coliform bacteria after filtrationand growth in TB for two and half hours Total cfu in Fold- 10 mlIncrease E. coli Input 92 cfu Control 2500 27.2 R1933-7/PEG (0.34 μm)1900 20.7 Isopore Polycarbonate filter (0.40 μm) 820 8.9 MF-MilliporeType HAWP (0.45 μm) 530 5.8 Enterobacter aerogenes Input 80 cfu Control2450 30.6 R1933-7/PEG (0.34 μm) 1500 18.8 Isopore Polycarbonate filter(0.40 μm) 900 11.3 MF-Millipore Type HAWP (0.45 μm) 450 5.6 Enterobactercloacae Input 52 cfu Control 1100 21.2 R1933-7/PEG (0.34 μm) 550 10.6Isopore Polycarbonate filter (0.40 μm) 310 6.0 MF-Millipore Type HAWP(0.45 μm) 170 3.3 Citrobacter braakii Input 10 cfu Control 150 15R1933-7/PEG (0.34 μm) 100 10 Isopore Polycarbonate filter (0.40 μm) 60 6MF-Millipore Type HAWP (0.45 μm) 40 4 Citrobacter freundii Input 80 cfuControl 900 11.3 R1933-7/PEG (0.34 μm) 650 8.1 Isopore Polycarbonatefilter (0.40 μm) 400 5.0 MF-Millipore Type HAWP (0.45 μm) 250 3.1

TABLE 17 Increase in cell number of coliform bacteria after filtrationand growth in TSB for three hours Total cfu Fold- in 10 ml Increase E.coli Input 88 cfu Control 5100 58.0 R1933-7/PEG (0.34 μm) 6000 68.2Isopore Polycarbonate filter (0.40μ m) 3500 39.8 MF-Millipore Type HAWP(0.45 μm) 2800 31.8 Enterobacter aerogenes Input 76 cfu Control 490064.5 R1933-7/PEG (0.34 μm) 4000 52.6 Isopore Polycarbonate filter (0.40μm) 2850 37.5 MF-Millipore Type HAWP (0.45 μm) 2200 28.9 Citrobacterfreundii Input 68 cfu Control 2050 30.1 R1933-7/PEG (0.34 μm) 1400 20.6Isopore Polycarbonate filter (0.40 μm) 700 10.3 MF-Millipore Type HAWP(0.45 μm) 875 12.9

Example 15 Development of Primers and Probes for Detection of E. coli byPCR

Two E. coli genes uidA (coding for b-glucoronidase) and tufA (coding forprotein chain elongation factor EF-Tu) were selected as target genes.PCR primers and probes were designed based on alignment of all thesequences available in GenBank. The primers designed were:

uidA: (SEQ ID NO: 1) Forward primer 5′-TCTACTTTACTGGCTTTGGTCG-3′(SEQ ID NO: 2) Reverse primer 5′-CGTAAGGGTAATGCGAGGTAC-3′ (SEQ ID NO: 3)Probe 5′-6-FAM-AGGATTCGATAACGTGCTGATGGTGC-  3′-Iowablack FQ tufA:(SEQ ID NO: 4) Forward primer: 5′-TCACCATCAACACTTCTCACG-3′(SEQ ID NO: 5) Reverse primer: 5′-CAGCAACTACCAGGATCGC-3′ (SEQ ID NO: 6)Probe: 5′-6-FAM-TGAATACGACACCCCGACCCG-3′-  Iowablack FQ

The primers and probes were synthesized by IDT DNA Technologies,Coralville, Iowa. Designed primers were used at 250 to 500 nM and probeat 125 to 250 nM with 10 μl 2× TaqMan Fast Universal Master Mix (AppliedBiosystems, Foster City, Calif.) and 5 μl of DNA template. In addition,commercially available reagents for detection of E. coli from PrimerDesign Ltd, Southampton, UK (Quantification of E. coli standard kit) andBioGx, Birmingham, Ala. (E. coli species Scorpions) were used accordingto manufacturer's instructions.

E. coli cells were diluted serially in Butterfield phosphate buffer andDNA template was prepared by mixing 100 ml of PREPMAN Ultra sample prepreagent (Applied Biosystems) with 25 μl of bacterial dilutions andboiling for 10 minutes. The boiled suspension was cooled, spun at 14,000RPM for 2 minutes and supernatant was transferred to a clean tube. 5 μlof DNA sample was added to 96-well PCR plate containing 20 μl ofreaction mix (primers, probes, and enzyme mix). Thermal cycling wascarried out using ABI 7500 sequence detection system with the followingconditions: 2 minutes at 95° C. for denaturation followed by 40 cyclesof: 20 seconds at 95° C. and 1 minute at 60° C. As shown below the limitof detection with the PCR was about 100 cfu.

TABLE 18 PCR detection of E. coli Approximate Concentration Primer ofbacteria in In-House reagents Design Kit BioGX kit PCR tube Ct Ct CtuidA tufA uidA NTC 40 40 40 40    1 cfu 40 40 40 40   10 cfu 38.34 39.3738.8 39.1   100 cfu 34.94 35.42 33.4 33.7  1000 cfu 30.96 30.33 29.829.5  10000 cfu 27.04 25.83 24.3 23.4 100000 cfu 22.25 22.86 20.3 20.7

Example 16 Detection of Bacteria from Spiked Water Samples by PCR

E. coli was grown over night in TSB at 37° C. The culture was diluted toobtain approximately 10 cfu/ml and 1 ml of the solution was added to1000 ml of sterile water to obtain approximately 10 cfu. This solutionwas filtered through various membranes at vacuum pressure of about 20inches of mercury using an AIR CADET Vacuum/Pressure Station. Themembranes were removed aseptically and added to a 50-ml tube with 10 mlof TB (Sigma-Aldrich Co., St. Louis, Mo.) and agitated at 300 rpm in aNewbrunswik Scientific shaker, Model Innova 4000 for two hours at 37° C.Control tubes were set up by spiking about 10 cfu (100 ml of 10² cfu/ml)into 10 ml TB and were grown similarly. All the samples were set up induplicates. At the end of two hours, growth media from one set of tubeswere plated on 3M PETRIFILM E. coli/Coliform Count Plates and incubatedovernight at 37° C. The plates were read using 3M PETRIFILM Plate Readerand colony forming units (cfu) were determined.

From the other set of tubes, the growth media containing cells were spunat 5000 rpm for 20 minutes to pellet cells. DNA was extracted usingQiagen Mini DNA extraction kit according to manufacturer's instructionsand DNA was eluted in 10 μl. 5 μl of extracted DNA was added to 20 μlPCR assay mix and PCR was carried out as described above with primer andprobes for uidA gene (Primer Design kit). The entire process fromfiltration followed by growth and detection by PCT took about 4 hours.

As shown below, the fold-increase varied from 5-fold to 18-fold. From1000 ml water samples spiked with 10 cfu of E. coli, the modified TIPSmembranes showed 9- to 18-fold increase and were positive by PCR. Thecommercial membranes showed only 5- to 6-fold increase and did not showany amplification of target DNA.

TABLE 19 Rapid Detection of E. coli by PCR Total cfu Fold- PCR AssayAmpli- in 10 ml increase (Ct) fication Input to 1000 ml water 10 cfu NTC40 No Control (no filtration) 156 15.6 32.1 Yes R1933-7/PEG (0.34 mm)150 15.0 32.7 Yes R1933-7/SPAN (0.34 mm) 175 17.5 31.9 Yes R1901-8B/PEG(0.51 mm) 98 9.8 34.2 Yes R1901-8B/SPAN (0.49 mm) 122 12.2 33.1 YesR1901-11/PEG (0.62 mm) 90 9.0 34.9 Yes R1901-11/SPAN (0.74 mm) 102 10.234 Yes PAN-3 (0.367 mm) 115 11.5 33 Yes Isopore Polycarbonate (0.40 mm)60 6.0 38.8 No MF-Millipore Type HAWP (0.45 mm) 50 5.0 39 No

Example 17 Preparation and Evaluation of Bags with PolypropyleneMembranes

A 4 wt % (weight %) surfactant solution was prepared by dissolvingsorbitol monolaurate (SPAN 20 available from Croda, New Castle Del.) in2-propanol (Alfa Aesar, Ward Hill, Mass.). R1930-10, R1901-11, andR1901-8B membranes (Table 1) were separately placed in polyethylene (PE)bags with sufficient surfactant solution to saturate them. The membranessaturated immediately. Excess surfactant solution was removed by rubbingthe bags to squeeze the solution out of the bag. The membranes wereremoved from the bags and air dried at room temperature. The propertiesfor the treated and untreated membranes were characterized for theproperties shown in Table 2. The Tight Zone Thickness refers to theapproximate thickness of the layer having the smaller pore size. Thedried membranes were stored in a plastic bag until used.

The membranes were constructed as a bag concentration device similar tothe device shown in FIGS. 11 and 12. Bags including each membrane wereconstructed in the same manner. An 8 inch by 8 inch ZIPLOC polyethylenebag (S.C. Johnson & Son, Inc., Racine, Wis.) was cut along the sealededges and separated into two pieces. A single layer of dry membrane wascut into a pentagonal shape having three square five-inch sides and twoequilateral sides of about 2.7 inches. The membrane was stacked atop apolypropylene nonwoven sheet (TYPAR, Reemay Inc, Charleston, S.C.)having the same dimensions with the open side of the multi-zone membranefacing the nonwoven sheet. The stack was then placed on the innersurface of one piece of the ZIPLOC bag with the square shape facing theZIPLOC seal and the triangle forming a v-shape near the bottom of thebag, and the tight side of the membrane facing the inner surface of thebag. FIG. 11 shows an exploded view of the bag concentration device 10with the following components: ZIPLOK bag sheets (container components12 a and 12 b with interlocking closure components 13 a and 13 b),polypropylene nonwoven sheet (nonwoven backing 20), filter membrane 14,and superabsorbent particles (absorbent member 16). Four edges of themembrane and nonwoven pentagon were heat sealed to the ZIPLOC bag toform a pouch with the five-inch side of the pentagon facing the ZIPLOCseal at the top of the bag left open, as shown in FIG. 12. The two sidesof the ZIPLOC bag were then heat sealed (seals 26) so the nonwoven facedthe opposing wall of the bag to form a concentration bag similar to theone shown in FIG. 8.

Each bag was evaluated by placing 3.8 g of superabsorbent hydro gelparticles (polyacrylate-polyalcohol) into each bag outside of the pouch.For each test, a broth was prepared by adding 1.125 ml of an ethyleneoxide/propylene oxide surfactant PLURONIC L64 (PL64, available fromBASF, Mount Olive, N.J.) and 0.45 g of bovine serum albumin (BSA,Sigma-Aldrich Co., St. Louis, Mo.) were added to 225 ml of sterilizedtryptic soy broth (TSB) obtained as Quick-Enrich TSB (3M Co., St. Paul,Minn.) to final concentrations of 0.5% PL64 and 0.2% BSA. The broth wasthen inoculated with 225 μl of Butterfield phosphate buffer containingapproximately 10⁵ cfu (colony forming units)/ml of Listeria innocua(ATCC 33090). The bacterial broth was then poured into the pouch of theconcentration bag (the tight side of the membrane) and propped uprighton a bench at room temperature until about three ml of the solutionremained in the pouch (20-30 minutes) and the absorption time wasrecorded. The remaining liquid was removed with a pipette andtransferred to a 15 ml graduated centrifuge tube to measure the volume.Each concentrated sample was then diluted 10-fold with Butterfield'sbuffer and 100 μl of the dilution was plated on a Modified Oxford Mediumplate (MOX plate obtained from Hardy Diagnostics, Santa Maria, Calif.)and incubated at 37° C. for 24 hours.

The control represents the initial concentration. Control samples wereprepared in the same manner as the evaluation assays but notconcentrated. Separate controls were tested with each set of membranes,e.g., Control 1 was tested at the same time as the SPAN 20 treatedmembranes and Control 2 was tested at the same time as the PEG treatedmembranes.

Each evaluation was replicated a second time and the BacteriaConcentration represents two different separate counts afterconcentrating. The concentration factor is the final concentrationdivided by the initial concentration of bacteria. Test results for theSPAN 20 treated membranes are shown in Table 20.

TABLE 20 Bacterial Recovery Rates and Absorption Times for TIPSMembranes Bacteria Volume Recovered concentration recovered BacteriaRecovery Concentration Absorption Membrane (cfu/ml) (ml) numbers ratefactor time (min) R1930-10 6850 3 2.06 × 10⁴ 58.9% 44 36 SPAN 20 57003.8 2.17 × 10⁴ 62.1% 37 36 R1901-11 9550 3 2.87 × 10⁴ 82.2% 62 22 SPAN20 7550 2.8 2.11 × 10⁴ 60.6% 49 22 R1901-8B 7850 3 2.36 × 10⁴ 67.5% 5120 SPAN 20 7900 3 2.37 × 10⁴ 68.0% 51 20 Control 1 155 225 3.49 × 10⁴ —— — R1901-8B 4250 23.4 1.45 × 10⁴ 75.6% 50 25 PEG 4050 3.2 1.30 × 10⁴67.8% 48 25 Control 2 69 225 1.55 × 10⁴ — — —

Example 18 Preparation and Evaluation of Bags with PolypropyleneMembranes

A 5 wt % stock solution of ethylene vinyl alcohol copolymer was preparedby dissolving an ethylene-vinyl alcohol copolymer (EVAL44) having 44mole % ethylene content (44% ethylene content poly(vinylalcoholco-ethylene polymer) obtained under the product number 414107 fromSigma-Aldrich Co., St. Louis, Mo.) in an ethanol (AAPER Alcohol andChemical Co. Shelbyville, Ky.)/water solvent mixture (70 vol % ethanol)in a water bath at temperature 70-80° C.

From the above stock solution, a polyethylene glycol (PEG) coatingsolution was made containing 1 wt % EVAL44, 2 wt % polyethylene glycol(600) diacrylate (obtained under product number SR610 from Sartomer,Warrington, Pa.), 1 wt % reactive photoinitiator VAZPIA(2-[4-(2-hydroxy-2-methylpropanoyl)phenoxy]ethyl-2-methyl-2-N-propenoylaminopropanoate, as disclosed in U.S. Pat. No. 5,506,279) in ethanol/watermixture solvent (70 vol % ethanol).

Microporous membranes R1933-7 and R1933-18 (Table 1) were saturated withthe PEG coating solution in a heavy weight PE bag, then removed from thebag. Excess solution was removed by wiping the surface of the saturatedmembrane with a paper towel. The membrane was air-dried at roomtemperature for 10-12 hours. The dry membrane was then saturated with a20 wt % NaCl aqueous solution and excess solution was removed. Themembrane was then placed on a conveyor belt of a UV curing system(Fusion UV system with H-bulb from Fusion UV Systems, Inc.,Gaithersburg, Md.) and cured in an inert nitrogen atmosphere chamber.The belt speed was 20 feet per minute (fpm). The membrane was turnedover and run through the UV system a second time at the same speed withthe opposite membrane side facing the light source. The cured membranewas washed in excess deionized water and dried at 90° C. for 1 to 2hours until completely dry. The dried membranes, with a permanenttreatment, were stored in a PE bag at room temperature.

Bag concentration devices were prepared as described in Example 17.

Example 19 Preparation and Evaluation of Bags with PolyacrylonitrileMembranes

Various polyacrylonitrile (PAN) polymer membranes (PAN-1, PAN-2, PAN-3,PAN-4, and PAN-5) were made as disclosed in Korean Patent ApplicationNo. KR20040040692. A 10.5-wt % solution of polyacrylonitrile (MW.150,000) polymer in N,N-dimethlyacetamide (DMAC) was prepared bydispersing the polymer in the liquid. A constant flow of PAN polymersolution (50 ul/min/hole) was pumped to a syringe connected to a highvoltage source. An electric force of 90-100 Kv was introduced to thesyringe which caused ejection of the polymer solution into the air toform electrospun PAN nanofibers. The fibers were collected on a web toform a bulky batt. To reduce the bulkiness and increase the structuralintegrity of this electrospun PAN nanofibers, post-treatment was carriedout by calendering at 140° C. and at pressures between about 10 to 20kgf/cm³. No other subsequent treatments were used. The membranes werestored in PE bags at room temperature. The membranes had pore sizes of0.2 μm, 0.53 μm, and 0.73 μm for the PAN-4, PAN-2, and PAN-5 membranes,respectively. Bag concentration devices were prepared as described inExample 17.

The bags were evaluated according to the same procedure as described inExample 17 except that 3.9 grams of hydrogel was used in each bag. Testresults are shown in Table 21.

TABLE 21 Bacterial Recovery Rate for Polyacrylonitrile MembranesBacteria Volume Recovered Membrane concentration recovered BacteriaRecovery Concentration Absorption (pore size) (cfu/ml) (ml) numbers ratefactor time (min) PAN-4 700 2.5 1.75 × 10³ 16.2% 15   40 (0.2 μm) 5003.5 1.75 × 10³ 16.2% 10   40 PAN-2 1850 3.7 6.85 × 10³ 63.4% 36 <10 (0.5μm) 2250 3.4 6.85 × 10³ 50.0% 47 <10 PAN-5 850 2.6 2..21 × 10³  20.5% 18<10 (0.7 μm) 1350 4.5 6.05 × 10³ 56.3% 28 <10 Control 48 225 1.08 × 10³— — —

Example 20 Preparation of Bags with Nylon Membranes

Nylon Membrane Liquid Filter product numbers 080ZN (0.8 μm) and 0606SN(0.6 μm) were obtained from 3M Purification Inc., Meriden, Conn.

Bag concentration devices were prepared as described in Example 17.

Example 21 Preparation of Bags with Polycarbonate Membranes

0.8 μm and 0.6 μm polycarbonate membrane filters were obtained from GEOsmonics (Hopkins, Minn.). Bag concentration devices were prepared asdescribed in Example 17.

Example 22 Preparation of Bags with Polyether/Polysulfone (PES)Membranes

0.8 μm and 0.6 μm polyether/polysulfone (PES) membranes were obtainedfrom GE Osmosics (Hopkins, Minn.). Bag concentration devices wereprepared as described in Example 17.

Example 23 Evaluation of Membranes

Bags containing a 0.6 μm nylon membrane (Example 20) and bags containinga 0.8 μm polycarbonate membrane (Example 21) were evaluated according tothe procedure described in Example 17 except that 4.0 grams of hydrogelwas used in each bag. Bags containing the 0.6 μm nylon membrane alsoevaluated using a TSB broth prepared as described above, except that thesurfactant used was 0.01% fluorosurfactant (3M NOVEC FC-4430, 3M Co.,St. Paul, Minn.) instead of PLURONIC L64. Test results are shown inTable 22.

TABLE 22 Bacteria Recovery From Nylon and Polycarbonate MembranesBacteria Volume Recovered Membrane concentration recovered BacteriaRecovery Concentration Absorption (pore size) (cfu/ml) (ml) numbers ratefactor time (min) Nylon 1600 2.3 3.60 × 10³ 17.8% 18 70 (0.6 μm) 16001.4 2.24 × 10³ 11.1% 18 70 Nylon 3200 1.6 5.12 × 10³ 25.3% 36 70 (0.6μm) 0.01% FC4430 Polycarbonate 1500 2.7 4.05 × 10³ 20.0% 17 90 (0.8 μm)2500 1.3 3.25 × 10³ 16.1% 28 90 Control 90 225 2.03 × 10⁴

Bags containing a 0.8 μm nylon membrane (Example 20) and bags containinga 0.8 μm PES membrane (Example 22) were evaluated according to theprocedure described in Example 17 except as follows. The amount ofhydrogel used in each bag was about 4.0 grams. The insides of the bagswere sterilized by spraying the inside of the bag with isopropylalcohol, propping the bag open, and irradiating the bag with ultravioletlight for 45 minutes. The tryptic soy broth contained 0.6% Yeast Extractprepared by dissolving 30 grams of TSB with 3 grams of Yeast Extract in1 liter of deionized water. One of two surfactants, as indicated inTable 23 was added to the broth—0.2% (w/v) PLURONIC L64 and 0.2% Tween80 (v/v) (Sigma-Aldrich Co., St. Louis, Mo.). The resulting broth (225ml) was inoculated with 225 μl of Butterfield's Buffer containing about1×10⁷ cfu/ml of Listeria innocua (ATCC 33090) and mixed well. The brothwas then poured into the pouch of the sample concentration bag andpropped upright for 50-90 minutes. The concentrated sample wascollected, measured, and diluted in Butterfield's buffer 10-foldsequentially in 3 ml. Then 1 ml of the 3^(rd) dilutions of each samplewas plated on AC PETRIFILM Plates (3M, Co. St. Paul, Minn.). The plateswere incubated at 37° C. for 24 hours and the colonies were counted.Table 23 shows the bacterial recovery results.

TABLE 23 Bacterial Recovery Rate for Polyether Sulfone and NylonMembranes Bacteria Volume Recovered Membrane concentration recoveredBacteria Recovery Concentration Absorption (Surfactant) (cfu/ml) (ml)numbers rate factor time (min) Nylon 0.8 μm 32000 6.5 2.08 × 10⁵ 26.0% 960 (0.2% Tween 20) Nylon 0.8 μm 396500 7.4 2.92 × 10⁵ 36.6% 11 60 (0.2%PL64) PES 29500 12.6 3.72 × 10⁵ 46.5% 8 100 (0.2% Tween 20) PES 345003.0 1.04 × 10⁵ 18.22% 10 100 (0.2% PL64) Control 3550 225 7.99 × 10⁴

Example 24 Large Water Sample Collection System with Float Valve andContainer

A large sample volume collection system was built using a four-gallonplastic carboy (P/N 073004, US Plastic Corporation, Lima Ohio). A 47 mmfilter holder (Cat # EW-06623-22, Cole-Parmer Instrument Co., VernonHills, Ill.) was attached to a ½″ float valve (Hudson Valve Company,Bakersfield, Calif.) using appropriate pipe fittings (Menards,Stillwater, Minn.). The float valve was fitted into the opening of thecarboy so that the float will rise to stop water flow when the requiredamount was reached. To the other end of the filter holder appropriatepipe fittings were attached so that the filter holder can be attached towater source. A 47 mm membrane filter was placed in the filter holderand the device was attached to water source (tap). The water source wasturned on and water was allowed to filter through the membrane. When thecontainer was full (10 liters), the float valve shut of the water flow.The tap was turned off, the filter holder was detached, and the membranefilter was removed and processed for further analysis. The membrane wasplaced on blood agar (Hardy Diagnostics, Santa Maria, Calif.) or trypticsoy agar plate (Hardy Diagnostics) and the plate was incubated at 37° C.for 16 to 24 hours. The colony forming units were counted to determinelevels of bacteria in 10 liters of water.

Example 25 Large Water Sample Collection System with Flow Meter

A large sample volume collection system was built by attaching a flowmeter (Cat. # WU-05610-01, Cole-Parmer Instrument Co., Vernon Hills,Ill.) to a 47 mm filter holder (Cat # EW-06623-22, Cole-Parmer) usingappropriate pipe fittings (Menards, Stillwater, Minn.). To the other endof the filter holder appropriate pipe fittings were attached so that thefilter holder can be attached to water source. A 47 mm membrane filterwas placed in the filter holder and the device was attached to watersource (tap). The water source was turned on and water was allowed tofilter through the membrane. The reading on the flow meter was used todetermine the amount of water flowing through the filter and when thewater flow reached the required amount (10 liters) the water flow wasshut off manually. The filter holder was detached and the membranefilter was removed and processed for further analysis. The membrane wasplaced on blood agar (Hardy Diagnostics, Santa Maria, Calif.) or trypticsoy agar plate (Hardy Diagnostics) and the plate was incubated at 37° C.for 16 to 24 hours. The colony forming units were counted to determinelevels of bacteria in 10 liters of water.

Example 26 Description of Process for Pipe Rehabilitation

1 to 10 liters of water sample is processed using preferred membranefilters. The retained bacteria can be eluted or grown further fordetection by assays such as PCR, isothermal amplification, immunoassays,etc. The rapid detection will enable to determine presence or absence ofbacteria in a short time (e.g., less than eight hours) allowing forfaster to return service of restored pipes.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference in their entirety. In theevent that any inconsistency exists between the disclosure of thepresent application and the disclosure(s) of any document incorporatedherein by reference, the disclosure of the present application shallgovern. The foregoing detailed description and examples have been givenfor clarity of understanding only. No unnecessary limitations are to beunderstood therefrom. The invention is not limited to the exact detailsshown and described, for variations obvious to one skilled in the artwill be included within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

Sequence Listing Free Text SEQ ID NO: 1 5′-TCTACTTTACTGGCTTTGGTCG-3′SEQ ID NO: 2 5′-CGTAAGGGTAATGCGAGGTAC-3′ SEQ ID NO: 35′-6-FAM-AGGATTCGATAACGTGCTGATGGTGC-3′- Iowablack FQ SEQ ID NO: 45′-TCACCATCAACACTTCTCACG-3′ SEQ ID NO: 5 5′-CAGCAACTACCAGGATCGC-3′SEQ ID NO: 6 5′-6-FAM-TGAATACGACACCCCGACCCG-3′- Iowablack FQ

1. A method comprising: passing a sample comprising at least onebiological organism through a filter membrane at a passive water volumeflux of at least 10 L/m²·h·psi, wherein the filter membrane comprises aBubble Point pore size of no more than 1.0 μm, thereby retaining atleast one biological organism on the surface of the membrane; anddetecting the at least one biological organism retained on the surfaceof the filter membrane.
 2. The method of claim 1 wherein the passivewater volume flux is at least 32 L/m²·h·psi.
 3. The method of claim 1wherein the passive water volume flux is at least 88 L/m²·h·psi.
 4. Themethod of claim 1 wherein the Bubble Point pore size is no more than 0.8μm.
 5. The method of claim 1 wherein the Bubble Point pore size is nomore than 0.2 μm.
 6. The method of claim 1 wherein at least 30% of thebiological organisms in the sample are retained on the surface of thefilter membrane.
 7. The method of claim 1 wherein at least 70% of thebiological organisms in the sample are retained on the surface of thefilter membrane.
 8. The method of claim 1 wherein at least 90% of thebiological organisms in the sample are retained on the surface of thefilter membrane.
 9. The method of claim 1 wherein at least 99% of thebiological organisms in the sample are retained on the surface of thefilter membrane.
 10. The method of claim 1 claim wherein at least 99.5%of the biological organisms in the sample are retained on the surface ofthe filter membrane.
 11. The method of claim 1 wherein the at least onebiological organism is detected in situ on the surface of the filtermembrane.
 12. The method of claim 1 wherein detecting the at least onebiological organism retained on the surface of the filter membranecomprises eluting the retained biological organism from the surface ofthe membrane prior to performing a detection assay.
 13. The method ofclaim 12 wherein at least 50% of the retained biological organisms areeluted from the surface of the filter membrane.
 14. The method of claim13 wherein at least 90% of the retained biological organisms are elutedfrom the surface of the filter membrane.
 15. The method of claim 1wherein detecting the at least one biological organism comprises atleast one of the following: contacting the biological organism with anantibody composition that specifically binds to the biological organism;detecting an enzyme activity of the biological organism; detecting abiological analyte of the biological organism; detecting a biologicalanalyte produced by the biological organism, followed by detecting aportion of a nucleic acid from the biological organism; amplifying atleast a portion of a nucleic acid molecule from the biological organism;and detecting the nucleotide sequence of the biological organism
 16. Themethod of claim 1 wherein the filter membrane comprises a polyolefinporous membrane, an ethylene-chlorotrifluoroethylene copolymer porousmembrane, a polyacrylonitrile porous membrane, a polycarbonate porousmembrane, a polyester porous membrane, a cellulose ester porousmembrane, a polyamide porous membrane, a polyethersulfone porousmembrane, a polysulfone porous membrane, a polyvinylidene fluoride(PVDF) porous membrane, a polyacrylonitrile nanofiber membrane, a PVDFnanofiber membrane, a cellulose ester nanofiber membrane, a polyvinylacetate or alcohol nanofiber membrane, or a polyvinyl butyral nanofibermembrane.
 17. The method of claim 1 wherein the filter membranecomprises a TIPS membrane or a nanofiber membrane.
 18. The method ofclaim 1 wherein the at least one biological organism is detected no morethan 24 hours after the sample is passed through the filter membrane.19. The method of claim 18 wherein the at least one biological organismis detected no more than 12 hours after the sample is passed through thefilter membrane.
 20. The method of claim 19 wherein the at least onebiological organism is detected no more than three hours after thesample is passed through the filter membrane.
 21. The method of claim 1wherein the sample comprises food.
 22. The method of claim 1 wherein thesample comprises an environmental sample.
 23. The method of claim 1wherein the sample comprises a water sample.
 24. The method of claim 1wherein the filter membrane is in functional communication with anabsorbent member.
 25. The method of claim 24 wherein the absorbentmember is enveloped by the filter membrane.
 26. The method of claim 1further comprising performing an assay to quantify the detectedbiological organisms recovered from the surface of the filter membrane27. The method of claim 1 further comprising: providing a device thatcomprises: a pocket comprising a pocket surface that defines a pocketvolume; an absorbent member disposed on at least a portion of the pocketsurface; and a filter membrane disposed on at least a portion of theabsorbent member in fluid communication with the pocket volume; whereinpassing a sample comprising at least one biological organism through afilter membrane comprises: adding the sample to the pocket volume;permitting the absorbent member to absorb at least 50% of the samplevolume.
 28. A filter device comprising: a pocket comprising a pocketsurface that defines a pocket volume; an absorbent member disposed on atleast a portion of the pocket surface; and a filter membrane disposed onat least a portion of the absorbent member in fluid communication withthe pocket volume.
 29. A filter device, comprising: a container; afilter membrane forming a pocket having an exterior surface, an interiorsurface, and an interior volume defined by the interior surface; anabsorbent member; wherein both the filter membrane and the absorbentmember are disposed in the container; wherein the absorbent member isdisposed in the container outside of the interior volume.
 30. The filterdevice of claim 29, wherein the container comprises a flexible,deformable container.
 31. The filter device of claim 29, wherein thepocket further comprises a nonwoven backing disposed adjacent theexterior surface of the filter membrane.
 32. The filter device of claim29, wherein the absorbent member is disposed on at least a portion ofthe exterior surface.
 33. The filter device of claim 29, furthercomprising a support member disposed in the container.
 34. The device ofclaim 29, wherein the pocket further comprises a sample port.